Requirement for self-recognition by T-helper cells and B cells has been analyzed in the in vitro primary antibody responses to the thymic dependent antigen TNP-KLH and the thymic independent antigen TNP-Ficoll. T cells obtained from A- greater than AxB, AxB- greater than A, and A-greater than B radiation bone marrow chimeras were tolerant to both A and B MHC determinants but could only cooperate with macrophages expressing the I-A determinants of the chimeric host, not the chimeric donor. The recognition by T-helper cells of macrophage I-A determinants was specifically blocked by anti-I-A antibodies directed at macrophage I-A determinants, suggesting that the I region gene products recognized by T-helper cells are also those detected serologically. Surprisingly, for TNP-Ficoll responses, B cells from AxB- greater than A and A-greater than B chimeras were also restricted to cooperating only with macrophages expressing MHC determinants of the chimeric host, strongly suggesting that B cell activation requires B cell recognition of macrophage MHC determinants.